Project Summary Patients with advanced and metastatic cancer have limited treatment options and face grim outcomes. There is an urgent need for novel and effective treatments. Hypoxia is a universal pathological feature of solid tumors. Hypoxic tumor cells acquire metastatic and lethal phenotypes primarily through the activities of hypoxia- inducible factor 1 alpha (HIF1?). Therefore, HIF1? is considered as a promising therapeutic target. To date, while several HIF1? inhibitors have been discovered and validated in preclinical studies, the clinical translations of these agents have been less successful. The robust antitumor effects seen in preclinical models are not observed in clinical trials. The reasons for the poor clinical efficacy are unclear. Thus, the long-term goal of this proposal is to understand how tumor cells adaptively respond and develop resistance to HIF1? inhibition, and harness this knowledge to translate HIF1? inhibitors into clinically effective and sustainable treatments. In preliminary studies, we specifically inhibited HIF1? using stable shRNA in multiple human tumor cell lines. We found that although tumor cells were initially sensitive to the antitumor activities of HIF1? inhibition, they quickly developed resistance in hypoxia in vitro and in xenografts in vivo even though HIF1? remained inhibited. Mechanistically, we observed that tumor cells adaptively responded to HIF1? inhibition by increasing the stability of oncogenic protein ID1 in hypoxia, and we found that silencing ID1 restored sensitivity to HIF1? inhibition, while overexpressing ID1 conferred resistance. In this proposal, we will test the hypothesis that HIF1? inhibition induces adaptive resistance via compensatory stabilization of oncogenic protein ID1 in hypoxia, which in turn supports the hypoxic growth of HIF1?-inhibited tumors. We further hypothesize that silencing ID1 or ID1-mediated oncogenic pathways will block the resistance and enhances the antitumor efficacy of HIF1? inhibition. We will use molecular biology and preclinical animal models to test this hypothesis in three specific aims. Aim 1: Determine the role of ID1 in conferring resistance to HIF1? inhibition in vitro and in vivo. Aim 2: Elucidate the mechanism by which ID1 protein is regulated by HIF1? in hypoxia. Aim 3: Determine the mechanism by which ID1 confers resistance to HIF1? inhibition in hypoxia. The successful completion of these aims may deepen our understanding of the role of HIF1? in cancer biology and therapy, may provide a rational approach to the clinical translation of HIF1?-inhibitory agents, and may lead to novel and lifesaving treatments for patients with advanced and metastatic disease.